Abstract
Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most inexpensive technologies available. Here, we describe four different protocols for uracil excision-based DNA editing: one for simple manipulations such as site-directed mutagenesis, one for plasmid-based multigene assembly in Escherichia coli, one for one-step assembly and integration of single or multiple genes into the genome, and a standardized assembly pipeline using benchmarked oligonucleotides for pathway assembly and multigene expression optimization.
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Cavaleiro, A.M., Nielsen, M.T., Kim, S.H., Seppälä, S., Nørholm, M.H.H. (2015). Uracil Excision for Assembly of Complex Pathways. In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols . Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_133
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DOI: https://doi.org/10.1007/8623_2015_133
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